Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: NOX4 regulates cell–cell adhesion in HCC cells. ( a ) NOX4 expression levels by quantitative PCR. Data represent the mean±s.e.m. ( N =4). ( b ) NOX4 and E-cadherin protein levels by western blot. β-actin was used as loading control. A representative experiment is shown ( N =3). Quantification normalized by loading control is shown under each blot. ( c ) Immunofluorescence of E-cadherin (green) and DAPI (blue; scale bar, 50 μm). ( d ) Representative bright-field images of cells cultured at basal conditions on top of plastic (left). Cell–cell contact imaging by immunofluorescence of E-cadherin (green) and ZO-1 (green); DAPI (blue; N =3; scale bar, 50 μm; right). ( e ) Analysis of CDH1 (E-cadherin) expression in PLC/PRF/5 cells by quantitative PCR. Data are mean±s.e.m. ( N =4). ( f ) E-cadherin protein levels by western blot. β-actin was used as loading control. A representative experiment is shown ( N =3). ( g ) Representative bright-field images of human HCC cells cultured on top of a bovine collagen I matrix (scale bar, 50 μm). ( h ) Quantification of the percentage of individual cells in each case. ( i ) Representative bright-field images of cells on top of a bovine collagen I matrix when NOX4 expression is silenced (scale bar, 50 μm). ( j ) Quantification of the percentage of individual cells in each case. ( k ) Number of cells per group. In ( h , j and k ) data are mean±s.e.m. ( N =4). See also .

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Cell Culture, Imaging

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: NOX4 suppresses the migratory/invasive potential of HCC cells. ( a ) Manual tracking of NOX4-silenced cells moving on a thick layer of collagen I/matrigel. Quantification of speed ( b ), distance ( c ) and directionality ( d ) of individual migrating cells. Data are expressed as mean±s.e.m. (20–30cells per condition). See also . Quantification of 3D invasion into a matrix of bovine collagen I (left) and collagen I/matrigel mix (1:1; right) of different human HCC cell lines ( e ) and of NOX4 knocked down Huh7 and PLC/PRF/5 cells ( f ). Data are expressed as fold increase versus Huh7 cells or shControl and are mean±s.e.m. ( N =3–5). ( g ) Invasive growth analysed embedding cells as spheroids in a bovine collagen I matrix. Representative images at day 0 and 4 (scale bar, 100 μm). ( h ) Representative images and quantification of the increase in area of the spheroids from control (shControl) and NOX4-silenced (shNOX4) cells between day 0 and 4 after embedding them into collagen (scale bar, 100 μm). ( i ) Percentage of HCC patients showing NOX4 deletion (23%) from the The Cancer Genome Atlas database ( N =249). Proportion of HCC patients in tumour grade 1/2 (17% left) and tumour grade 3/4 (32% right) showing NOX4 deletion.

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques:

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: NOX4 suppresses actomyosin contractility in HCC cells. In all experiments cells were cultured on top of a bovine collagen I matrix. ( a and d ) Percentage of cells with blebs. ( b and e ) quantification of phosphorylated MLC2 (pMLC2) immunostaining intensity per cell surface. ( c and f ) Representative confocal images of pMLC2 (green), F-actin (red) and DAPI (blue) staining ( N =4; scale bar, 20 μm, square with a higher magnification is shown for better visualisation). White arrows indicate blebs. Data in ( a , b , d and e ) are mean±s.e.m. ( N =4, at least five pictures per experiment). ( g ) Phosphorylated and total MLC2 protein levels. GAPDH was used as loading control. A representative experiment is shown ( N =3). ( h ) 3D imaging of PLC/PRF/5 (shControl and shNOX4) and SNU449 cells invading a collagen I/matrigel mix (1:1) matrix. Higher magnification is shown for better visualization. Black arrow indicates direction of invasion.

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques: Cell Culture, Immunostaining, Staining, Imaging

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: Overexpression of NOX4 in SNU449 cells switches to a parenchymal phenotype, and suppresses actomyosin contractility and invasive behaviour. ( a ) NOX4 mRNA (up) and protein (down) levels in SNU449 cells overexpressing NOX4 (+NOX4) and in Huh7 cells. Beta-actin used as a loading control. ( b ) ZO-1 (green) and Vinculin (green) immunofluorescence. DAPI (blue) to detect nuclei ( N =3; scale bar, 50 μm). ( c ) Representative confocal images of immunostaining of pMLC2 and F-actin of HCC cells cultured on top of bovine collagen I matrix ( N =3; scale bar, 50 μm). ( d ) Phosphorylated and total MLC2 protein levels. GAPDH was used as loading control. A representative experiment is shown ( N =3). ( e ) Quantification of pMLC2 immunostaining intensity per cell surface. ( f ) Quantification of the percentage of individual cells cultured on top of a collagen I matrix. ( g ) Manual tracking of SNU449 cells moving on a thick layer of collagen I/matrigel. Quantification of speed ( h ) and distance ( i ) of individual migrating cells. Data are mean±s.e.m. (20–30cells per condition). ( j ) Quantification of 3D invasion into a bovine collagen I matrix. ( k ) Representative images of spheroids from control and NOX4 overexpressing cells at day 0 and 2 after embedding them into collagen I (scale bar, 100 μm). ( l ) Quantification of the increase in area of the spheroids between day 0 and 2. Data in ( a , c , f , j and l) are mean±s.e.m. ( N =3).

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques: Over Expression, Immunofluorescence, Immunostaining, Cell Culture